Download Slides

The watchmaker's apprentice:
Building a synthetic genetic oscillator
with parts borrowed from nature
Mukund Thattai
Simons Centre for the Study of Living Machines
NCBS/TIFR
September 2013
The blind watchmaker
generates complexity through
the undirected processes of
mutation and selection...
The blind watchmaker
generates complexity through
the undirected processes of
mutation and selection...
... while the watchmaker's apprentice
has a design in mind, and uses the
complex parts invented by nature to
realize it
A brief introduction to
genetic networks
The E. coli genome contains about 4000 genes...
1µM
... and the human genome contains about 24000 genes
Genes can be turned on and off, allowing
combinatorial complexity
protein
protein
mRNA
mRNA
RNAP
DNA
promoter
gene
DNA
promoter
gene
Genes can be turned on and off, allowing
combinatorial complexity
protein
mRNA
activator
RNAP
protein
mRNA
inhibitor
RNAP
DNA
promoter
gene
DNA
promoter
gene
Genes can be connected into networks with
useful dynamical properties
Noise reduction
Memory
Oscillations
Negative feedback
Positive feedback
Hysteretic oscillator
Flip-flop
Ring oscillator
We can build networks from basic parts...!
Inducers:!
Off-the-shelf chemicals!
Reporters:!
Fluorescent proteins!
Transcriptional regulators:!
DNA-binding proteins!
Promoters:!
DNA segments that control
gene expression!
The host strain:!
Escherichia coli!
... but getting things to work is hard!!
In principle, UV exposure
would trigger transient
expression of lacY, and the
re s u l t i n g T M G u p t a k e
would turn on lac
expression. In practice, the
design did not function as
expected. We soon realised
that the presence of
extraneous lacI binding sites
on the plasmid backbone
had perturbed the system.
However, imple- menting such a
gradient turned out to be far
from trivial. Moreover, we were
not able to rescue chemotaxis in
the knockout strain.
As expected, the RFP control
signal did not oscillate. However,
synchronisation could not be
achieved because the presence of
DnaA boxes did not seem to
affect cell-cycle progression.
& S. Dabholkar, M. Thattai. Brainstorming biology. IET Synth. Biol. 1, 17-20 (2007)
... but getting things to work is hard!!
In principle, UV exposure
would trigger transient
expression of lacY, and the
re s u l t i n g T M G u p t a k e
would turn on lac
expression. In practice, the
design did not function as
expected. We soon realised
that the presence of
extraneous lacI binding sites
on the plasmid backbone
had perturbed the system.
However, imple- menting such a
gradient turned out to be far
from trivial. Moreover, we were
not able to rescue chemotaxis in
the knockout strain.
As expected, the RFP control
signal did not oscillate. However,
synchronisation could not be
achieved because the presence of
DnaA boxes did not seem to
affect cell-cycle progression.
& S. Dabholkar, M. Thattai. Brainstorming biology. IET Synth. Biol. 1, 17-20 (2007)
Negative feedback
Noise reduction
X
PLtetO1
tetR*-egfp
PLtetO1
tetR*-egfp
& Figures adapted from Hasty et al, 2002
& Becskei & Serrano, Nature, 2000
Flip flop
Memory
IPTG!
PLs1con!
lacI!
& Figures adapted from Hasty et al, 2002
cIts!
gfp!
Ptrc-2!
Heat!
& Gardner et al., Nature, 2000
Positive feedback
Memory
TMG
Plac
lacY
Plac
gfp
& Ozbudak & Thattai et al., Nature, 2004
Ring oscillator
Oscillations
PLtetO1
gfp
PLtetO1
cI
Plac
lacI
tetR
PR
& Figures adapted from Hasty et al, 2002
& Elowitz & Leibler, Nature, 2000
Hysteretic oscillator
Synchronized oscillations
luxI::cfp
Plac
lacI
PR
PR
luxR::yfp
& Rai et al., PLoS Comp Biol, 2012
The basic parts:
Borrowing from a cell
communication system
Bacteria can change their behaviour
depending on local cell density!
Density-dependent
bioluminescence in
Vibrio harveyi!
Symbiosis between
bioluminescent Vibrio fischeri
and the hawaiian bobtail squid!
& MJ McFall-Ngai & EG Ruby, University of Hawaii!
& F Ausubel, MGH!
& MB Miller & BL Bassler, 2001!
Quorum sensing during biofilm
formation in Pseudomonas!
aeruginosa
“Quorum sensing” is a form of
chemical cell-to-cell communication!
receiver
output!
cell density (ρ)!
issuer
signal
Quorum-sensing systems are built from a
handful of conserved molecular components!
signal
issuer (I)
receiver (R)
AHL!
I!
R!
xρ
pX!
pR!
R!
R!
pR!
& Gray and Garey, 2001; Vannini et al., 2002
Output!
A topological puzzle:!
All characterized natural QS systems use I-feedback!
Species:!
Niche:!
Process:!
Vibrio fischeri!
marine endosymbiont!
bioluminescence!
glucose!
LuxI!
AHL!
LuxR!
Pseudomonas aeruginosa!
human pathogen!
virulence; biofilm formation!
Agrobacterium tumefaciens!
plant pathogen!
plasmid conjugation!
signalling!
octopine!
TraI!
TraR!
LasI!
AHL!
LasR!
AHL1!
RhlI!
AHL2!
RhlR!
glucose!
LuxI!
LuxR!
AHL!
What is happening inside the biochemical black box?!
!
What is the role of transcriptional feedback?!
!
Why is one feedback topology preferred over another?!
Inferring feedback behavior from feedforward data!
Feedforward!
I!
R!
pR!
1 dYZ
= QZ f (µρYI ,YR ) − YZ
γ Z dt
extract biochemistry!
(two regulated inputs)!
Z!
α
R-feedback!
I!
R!
pR!
1 dYR
= QR f (µρYI ,YR ) − YR
γ R dt
α
I-feedback!
I!
R!
pR!
1 dYI
= QI f (µρYI ,YR ) − YI
γ I dt
predict
feedback
responses!
(single
regulated
input)!
& Rai et al., PLoS Comp Biol, 2012
Inferring feedback behavior from feedforward data!
Extracting biochemistry: the feedforward response!
aTc!
IPTG!
pT!
pL!
I!
R!
pR!
CFP!
log10(CFP)
& Rai et al., PLoS Comp Biol, 2012
The 2-D promoter logic function of pR!
f (µρYI ,YR ) =
log10(CFP)
 YI )m )
YRn (δ + (µρ
β + (1− β )
 YI )m )
1+ YRn (δ + (µρ
n: Hill coefficient of
LuxR-DNA binding!
5 parameter fit to data!
& Thattai, Phil. Trans. Roy. Soc. A, 2012
R-feedback response: monostable, smooth!
α
α
Regulator!
log10(CFP)
I!
Input!
R!
pR!
CFP!
Output!
α
Output!
Regulator!
I!
R!
pR!
α
Output!
& Rai et al., PLoS Comp Biol, 2012
I-feedback response: hysteretic, switch-like!
α
Input!
log10(CFP)
R!
pR!
CFP!
Output!
α
Regulator!
Output!
α
I!
Regulator!
I!
R!
pR!
α
Output!
& Rai et al., PLoS Comp Biol, 2012
Phase diagram of density-dependent responses!
α
α
n
Output!
n
& Rai et al., PLoS Comp Biol, 2012
Endgame:
Building an oscillator from
the bottom up
The “hysteretic oscillator” motif:!
negative feedback with delay!
Drosophila circadian clock
12h dark
activity
Zebrafish segmentation clock
12h light
lights out
[synchronized]
& Gallego & Virshup, Nat. Rev. Mol. Cell Biol., 2007
& Panda et al., Nature, 2002!
& Horikawa et al., Nature, 2006!
Synthetic oscillators!
& Atkinson et al., Cell, 2003!
& Stricker et al., Nature, 2008!
& Danino et al., Nature, 2010!
[synchronized]
Oscillator protein dynamics!
Roadmap: putting parts together to make the oscillator!
I!
R!
pR!
Z!
2 inputs
1 input
0 inputs
Experiments: The synchronization protocol !
Nitrogen-limited chemostat
Input
Output:
minimal medium
OD600 [cell density]
limiting NH4Cl
± AHL
imaging CFP/YFP
Control: continuous AHL
measure
Test: Synchronize with 12h AHL
measure
Experiments: Synchronized oscillations over 24h!
measure
measure
measure
measure
6 Herioc experiments by Navneet Rai!
Promoter logic explains oscillations in dual feedback system!
The regulation / biochemistry / topology hierarchy!
seconds
102
Software:!
Regulation!
104
106
Firmware:!
Biochemistry!
108
1010
Hardware:!
Topology!
1012
Acknowledgements!
!
Navneet Rai, Rajat Anand, Sugat Dabholkar!
KV Venkatesh (ChemE, IIT Bombay)!
!
!
Undergraduates:!
Krishna Ramkumar (IIT Bombay), Sushant More (IISER Pune),!
Varun Sreenivasan (St. Xavier’s Bombay), Shashanka Kundu (St.
Stephen’s), Vini Gautam (Delhi University), Senthil Kumar (PSG
Coimbatore)!
!
!
Further reading:!
> Rai, N. et al., Prediction by promoter logic in bacterial quorum
sensing. PLoS Comp. Biol. 8, e1002361 (2012).!
> Thattai, M. Using topology to tame the complex biochemistry of
genetic networks. Phil. Trans. Roy. Soc. A 20110548 (2012).!
[email protected] !